Fatty acids are important components constituting lipids such as phospholipid and triacylglycerol. Fatty acids having two or more unsaturated bond sites are collectively called polyunsaturated fatty acids (PUFAs). Specifically, for example, arachidonic acid, dihomo-γ-linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid are known, and various bioactivities thereof have been reported (Non-Patent Literature 1). Some of the polyunsaturated fatty acids cannot be synthesized in animal bodies, and such polyunsaturated fatty acids should be ingested from foods as essential fatty acids.
In animal bodies, the polyunsaturated fatty acids are contained in various organs and tissues. For example, arachidonic acid is isolated from lipids extracted from suprarenal gland and liver of animals. The amounts of these polyunsaturated fatty acids contained in animal organs are, however, small, and the polyunsaturated fatty acids extracted and isolated from animal organs only are insufficient for a large amount of supply thereof. Thus, microbial techniques have been developed for obtaining polyunsaturated fatty acids by culturing various microorganisms. In particular, microorganisms in the genera Mortierella are known to efficiently produce lipids containing polyunsaturated fatty acids such as arachidonic acid. Other attempts have also been made to produce polyunsaturated fatty acids in plants. Polyunsaturated fatty acids are known to constitute reserve lipids such as triacylglycerol and accumulate within microorganism cells or plant seeds.
Triacylglycerol as a reserve lipid is generated in living bodies as follows: An acyl group is transferred to glycerol 3-phosphate by glycerol 3-phosphate acyltransferase to generate lysophosphatidic acid. Another acyl group is transferred to the lysophosphatidic acid by lysophosphatidic acid acyltransferase to generate phosphatidic acid. The phosphatidic acid is dephosphorylated by phosphatidic acid phosphatase to generate diacylglycerol. A further acyl group is transferred to the diacylglycerol by diacylglycerol acyltransferase to ultimately generate triacylglycerol.
It is known that in the triacylglycerol biosynthetic pathway or the phospholipid biosynthetic pathway mentioned above, glycerol 3-phosphate acyltransferase (hereinafter, also referred to as “GPAT”: EC 2.3.1.15) involves a reaction generating lysophosphatidic acid through acylation of glycerol 3-phosphate.
Existence of a GPAT gene has been reported in some organisms. As GPAT genes derived from mammals, two types of GPAT genes, i.e., a microsomal type (membrane-bound form) and a mitochondrial type (membrane-bound form), have been cloned (Non-Patent Literature 2). As GPAT genes derived from plants, three types of GPAT genes, i.e., a microsomal type (membrane-bound form), a mitochondrial type (membrane-bound form), and a chloroplast type (free form), have been cloned (Non-Patent Literature 3).
As GPAT genes derived from fungi, Saccharomyces cerevisiae, two types of GPAT genes, i.e., microsomal type (membrane-bound form) GPT2/GAT1 (YKR067w) and SCT1/GAT2 (YBL011w), have been cloned, and it is known that simultaneous deletion of these types of GPAT genes results in death (Non-Patent Literature 4). It has been shown that GPT2 has an activity showing broad substrate specificity to fatty acids from palmitic acid (16:0) to oleic acid (18:1), whereas SCT1 shows high substrate selectivity to fatty acids having 16 carbon atoms such as palmitic acid (16:0) and palmitoleic acid (16:1) (Non-Patent Literature 4).
In addition, the GPAT gene has been cloned from various biological species. In particular, GPAT derived from a lipid-producing fungus, the genera Mortierella, is reported as follows.
Regarding GPAT derived from Mortierella ramanniana, a microsomal type GPAT has been isolated, and it has been shown that this GPAT preferentially uses oleic acid (18:1) as an acyl donor with a selectivity as 5.4 times high as that to palmitic acid (16:0) (Non-Patent Literature 5). Regarding GPAT derived from Mortierella alpina (hereinafter, also referred to as “M. alpina”), it has been reported that a glycerol 3-phosphate acyltransferase activity resides in a microsomal fraction (Non-Patent Literature 6).
It has been shown that, when GPAT (membrane-bound form) present in microsome of M. alpina is reacted with various acyl-CoAs in vitro, the GPAT uses a broad range of polyunsaturated fatty acids, such as oleic acid (18:1), linoleic acid (18:2), dihomo-γ-linolenic acid (DGLA) (20:3), and arachidonic acid (20:4), as substrates, with maitaining its high activity (Patent Literature 1).
It has been shown that GPAT cloned from M. alpina (ATCC No. 16266) (hereinafter, referred to as MaGPAT1 (ATCC No. 16266)) was expressed in Yarrowia lipolytica that had been transformed such that eicosapentaenoic acid (EPA) can be biosynthesized, and as a result, a proportion of dihomo-γ-linolenic acid (DGLA) (20:3) increased, whereas a proportion of oleic acid (18:1) decreased, among the total fatty acids. This demonstrated that a polyunsaturated fatty acid having a longer chain and a higher degree of unsaturation was selectively incorporated (Patent Literature 2).
In recent studies, a GPAT homolog, MaGPAT2, was isolated from M. alpina (strain 1S-4), and it has been reported that the homolog has a substrate specificity different from that of MaGPAT1 (Patent Literature 3). That is, when they are expressed in yeast, MaGPAT1 increases the content of palmitic acid in the lipid produced by the yeast, whereas MaGPAT2 increases the content of oleic acid in the lipid produced by the yeast.